Synthetic nucleic acid microarrays have gained tremendous importance in the analysis of nucleic acid samples from biological sources. They are powerful tools that are suitable for the analysis of complex mixtures of nucleic acids as required in the biological and biomedical sciences. Applications of microarrays include research with the aim to understand the correlation of gene sequences with their function, the analysis of the expression pattern of various organisms and tissues that can be employed in drug discovery, the analysis of genetic variation that occurs between individuals, which is employed in the development of individual medicines, and the analysis of mutation patterns that occur in specific tissues, which is useful in cancer research and diagnosis. Another application of is the use of microarrays to sequence nucleic acids by the analysis of the hybridization pattern obtained from a particular nucleic acid sequence on an array. As almost all analysis techniques that are based on nucleic acid hybridization can be performed using nucleic acid microarrays there are numerous other important applications of the microarrays and new applications continue to emerge.
In applications of nucleic acid microarrays, the surface bound oligonucleotides of the arrays (probes) are used to analyze sample sequences (targets) through complementary recognition (hybridization) in a parallel fashion. Driven by the complexity of the nucleic acid sequence information in biological samples and the value of the usually small biological samples there is an increasing demand for microarrays with miniaturized features that allow a highly parallel analysis of such small samples. This demand is best met through the light-directed synthesis of microarrays for the parallel, in situ assembly of oligopeptides and oligonucleotides on supports as originally described by e.g., Fodor et al. (1991) Science 251:767-773; Pirrung et al., U.S. Pat. No. 5,143,854; and Chee et al. (1996) Science 274:610-614. Due to its rapid advancement to date this approach allows the construction of feature sizes of 2.1×10−4 μm2 and possibly even smaller, as described by Singh-Gasson et al. (1999) Nature Biotech. 17:974-978. In contrast, microarrays that are assembled by spotting prefabricated oligonucleotides or cDNA samples onto surfaces allow the fabrication of feature sizes of about 75 μm2, as reviewed for example in Bowtell and Sambrook, eds., “DNA Microarrays: A Molecular Cloning Manual,” Cold Spring Harbor Laboratory Press (2002). Due to the easy and flexible spatial addressing of reaction sites on the support, a further advantage of light-directed array assembly is that it can be performed in a parallel and automated fashion.
In order to become more widely used microarrays must be available at reasonable cost, which is driven by various factors including the consumption of reagents in their manufacturing, the degree of automation employed and the throughput of manufactured arrays obtained in the production set-up. Since microarrays from light directed synthesis are built sequentially on the array surface by a multitude of chemical processes, it is of utmost importance to optimize the individual steps employed in order to increase the throughput.
Light-directed syntheses of microarrays largely rely on the conventional, highly optimized schemes for the synthesis of oligonucleotides on solid supports exploiting the phosphoramidite chemistry as described e.g. by Beaucage et al. (1992) Tetrahedron 48:2223-2311, which is incorporated herein by reference in its entirety. They comprise the stepwise attachment of nucleoside synthons activated by a phosphoramidite group, in a predetermined order to either, depending on the direction of chain extension, the 5′-functional group or the 3′-functional group of the growing strand, which is linked to a support such as the surface of a DNA chip. Each elongation step usually consists of a reaction cycle including the deprotection of either the 5′- or the 3′-hydroxyl group of the growing strand, chain extension by addition of a nucleoside phosphoramidite and an activator, the optional capping of unreacted terminal hydroxyl groups, and the oxidation of the newly formed internucleosidic phosphorous linkage to the pentavalent state. Arrays comprising oligonucleotide probes assembled in the 5′ to 3′ direction are suitable for assays relying on hybridization, as well as assays involving enzymatic reactions, e.g. elongation or ligation, since the 3′-ends of its oligonucleotide probes are freely accessible, as described by Beier et al. (2001) Helv. Chim. Acta 84:2089-2095.
Since all of the reactions of a chain extension cycle, apart from the deprotection step, are basically carried out according to well-elaborated conventional methods and involve the use of vast excesses of reagents, they are essentially quantitative. In the light-directed microarray assembly of oligonucleotides, the step of removing the standard acid-labile dimethoxytrityl (DMT) protective group is replaced by the photochemical removal of a light sensitive protective groups, which is the limiting reaction with respect to the efficiency and duration of the elongation cycle. Thus, for the commonly used [(α-methyl-2-nitropiperonyl)-oxy]carbonyl (MeNPOC) protective group the yield of the photochemical deprotection on the support in the “dry” mode, i.e. without solvent, is only about 90%, resulting in a cycle efficiency of less than 90% as described by Barone et al. (2001) Nucleosides, Nucleotides & Nucleic Acids 20:525-531 and McGall et al. (1997) J. Am. Chem. Soc. 119:5081-5090, each of which is incorporated herein by reference in its entirety. Such conversion rates are rather moderate compared to the standard technology employing DMT-protection, leading to the formation of failure sequences and to truncated oligonucleotide probes, with the desired full-length sequences representing in case of an array with 20-mer probes only about 10% of the total.
Furthermore, removal of the MeNPOC group in the dry state requires irradiation times of about 1 minute or longer if the deprotection is performed in the presence of a solvent. For each elongation step in the synthesis of an oligonucleotide array four photolytic steps are necessary, corresponding to the coupling steps, which are subsequently performed with each of the four nucleotidic synthons in pre-defined areas on the support. The total time needed for deprotection adds up to 4×N minutes for the assembly of an array of N-mers. Thus, in order to achieve higher throughput rates of array assembly short deprotection times are crucial.
The photocleavage of MeNPOC-protected nucleosides proceeds most rapidly under dry conditions or in less polar solvents, such as toluene or dioxane. Additionally, the resulting yields and the corresponding coupling efficiencies are moderate at most as discussed above. In contrast, the also widely-used 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) group requires irradiation times that are significantly longer in a dry state, but much shorter in polar solutions, such as mixtures of water with acetonitrile or methanol, as described by Giegrich et al. (1998) Nucleosides & Nucleotides 17:1987-1996. Further enhanced deprotection rates for NPPOC-nucleosides were accomplished by Beier et al. (2000) Nucleic Acids Res. 28:e11, via irradiation in acetonitrile containing a small amount of a base, such as DBU or NMI. This method provides up to 12% higher cycle efficiency than the application of MeNPOC-protected phosphoramidites in a dry deprotection format.
Early reports on photocleavable protective groups in nucleoside and nucleotide chemistry were related to the ethers and carbonate esters of the ortho-nitrobenzyl group including derivatives thereof, as described by Pillai, in Organic Photochemistry, Ed. Padwa, Marcel Dekker, NY and Basel, 1987, Vol. 9, p. 225-323 and Walker et al. (1988) J. Am. Chem. Soc. 110:7170-7177. The MeNPOC protective group can be considered as an advanced variant in this category.
Pyrenylmethoxycarbonyl-type protective groups have also been used as described by McGall and Rava, International Patent Application Publication No. WO 98/39348. The respective nucleoside and nucleotide derivatives of the aforementioned protective groups, although resulting in arrays of sufficient quality for certain applications, have unsatisfactory deprotection rates and yields. Additionally, upon cleavage of the ortho-nitrobenzyl compounds undesired side products like toxic nitrosophenyl compounds are formed.
Enhanced cleavage rates and/or reduced levels of side products have been observed for dimethoxybenzoin carbonates as described by Pirrung and Bradley (1995) J. Org. Chem. 60:1116-1117, and for both ortho-nitrophenylethyl-type carbonates, such as the above described NPPOC, and ortho-nitrophenylethyl-type sulfonates as described by Hasan et al. (1997) Tetrahedron 53:4247-4264; Giegrich et al. (1998) Nucleosides & Nucleotides 17:1987-1996; and Pfleiderer et al., U.S. Pat. Nos. 5,763,599 and 6,153,744. The former photolabile group displays faster deprotection rates, whereas the two latter groups also display higher deprotection yields and purities. However, as discussed above, even these “second generation” photolabile protective groups there remains a need for improvement with respect to faster deprotection and higher conversion rates.
In the light of an increasing demand methods have been devised using digital light processors to manufacture gene chips having any individual design within hours as described e.g. by Singh-Gasson et al. (1999) Nature Biotech. 17:974-978. The overall throughput of such instruments, however, is currently limited to about two chips per working day, mainly due to the extensive amount of accumulated deprotection time in an array synthesis. Thus, speeding up the photolysis time would significantly cut the overall process time.
The present invention provides novel photolabile protective groups, which are well suited for both the 3′-OH— and 5′-OH function of the sugar moiety of nucleoside derivatives. The novel protective groups have improved deprotection properties allowing for significantly accelerated array assembly and enhanced oligonucleotide quality. Furthermore, the protective groups are specifically adapted to ‘dry’ or ‘wet’ deprotection conditions in order to allow high-throughput and high-quality array fabrication independent of the deprotection approach used.